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Fungal fermentation of food and agricultural by-products holds promise for improving food sustainability and security. However, the molecular basis of fungal waste-to-food upcycling remains poorly understood. Here we use a multi-omics approach to characterize oncom, a fermented food traditionally produced from soymilk by-products in Java, Indonesia. Metagenomic sequencing of samples from small-scale producers in Western Java indicated that the fungus Neurospora intermedia dominates oncom. Further transcriptomic, metabolomic and phylogenomic analysis revealed that oncom-derived N. intermedia utilizes pectin and cellulose degradation during fermentation and belongs to a genetically distinct subpopulation associated with human-generated by-products. Finally, we found that N. intermedia grew on diverse by-products such as fruit and vegetable pomace and plant-based milk waste, did not encode mycotoxins, and could create foods that were positively perceived by consumers outside Indonesia. These results showcase the traditional significance and future potential of fungal fermentation for creating delicious and nutritious foods from readily available by-products.

The article considers the distribution and mechanisms of the meiotic drive as a phenomenon manifested in unequal transmission of gene alleles and/or homologous chromosomes into gametes during meiosis. The meiotic drive has been studied in the most detail in Drosophila, mice, corn, and ascomycete fungi of the genera Neurospora and Podospora. The consequence of the meiotic drive is a shift in the frequencies of alleles in the gene pool and the maintenance of nonadaptive traits in the population. [ABSTRACT FROM AUTHOR]

Advances in genomics and transcriptomics accompanying the rapid accumulation of omics data have provided new tools that have transformed and expanded the traditional concepts of model fungi. Evolutionary genomics and transcriptomics have flourished with the use of classical and newer fungal models that facilitate the study of diverse topics encompassing fungal biology and development. Technological advances have also created the opportunity to obtain and mine large datasets. One such continuously growing dataset is that of the Sordariomycetes, which exhibit a richness of species, ecological diversity, economic importance, and a profound research history on amenable models. Currently, 3,574 species of this class have been sequenced, comprising nearly one-third of the available ascomycete genomes. Among these genomes, multiple representatives of the model genera Fusarium, Neurospora, and Trichoderma are present. In this review, we examine recently published studies and data on the Sordariomycetes that have contributed novel insights to the field of fungal evolution via integrative analyses of the genetic, pathogenic, and other biological characteristics of the fungi. Some of these studies applied ancestral state analysis of gene expression among divergent lineages to infer regulatory network models, identify key genetic elements in fungal sexual development, and investigate the regulation of conidial germination and secondary metabolism. Such multispecies investigations address challenges in the study of fungal evolutionary genomics derived from studies that are often based on limited model genomes and that primarily focus on the aspects of biology driven by knowledge drawn from a few model species. Rapidly accumulating information and expanding capabilities for systems biological analysis of Big Data are setting the stage for the expansion of the concept of model systems from unitary taxonomic species/genera to inclusive clusters of well-studied models that can facilitate both the in-depth study of specific lineages and also investigation of trait diversity across lineages. The Sordariomycetes class, in particular, offers abundant omics data and a large and active global research community. As such, the Sordariomycetes can form a core omics clade, providing a blueprint for the expansion of our knowledge of evolution at the genomic scale in the exciting era of Big Data and artificial intelligence, and serving as a reference for the future analysis of different taxonomic levels within the fungal kingdom. [ABSTRACT FROM AUTHOR]

Functional crosstalk between DNA methylation, histone H3 lysine-9 trimethylation (H3K9me3) and heterochromatin protein 1 (HP1) is essential for proper heterochromatin assembly and genome stability. However, how repressive chromatin cues guide DNA methyltransferases for region-specific DNA methylation remains largely unknown. Here, we report structure-function characterizations of DNA methyltransferase Defective-In-Methylation-2 (DIM2) in Neurospora. The DNA methylation activity of DIM2 requires the presence of both H3K9me3 and HP1. Our structural study reveals a bipartite DIM2-HP1 interaction, leading to a disorder-to-order transition of the DIM2 target-recognition domain that is essential for substrate binding. Furthermore, the structure of DIM2-HP1-H3K9me3-DNA complex reveals a substrate-binding mechanism distinct from that for its mammalian orthologue DNMT1. In addition, the dual recognition of H3K9me3 peptide by the DIM2 RFTS and BAH1 domains allosterically impacts the DIM2-substrate binding, thereby controlling DIM2-mediated DNA methylation. Together, this study uncovers how multiple heterochromatin factors coordinately orchestrate an activity-switching mechanism for region-specific DNA methylation. DIM2-mediated DNA methylation contributes to heterochromatin formation in Neurospora. This study identifies that HP1 protein and the H3K9me3 mark synergistically control DIM2 activity, providing a mechanism for heterochromatin-guided DNA methylation. [ABSTRACT FROM AUTHOR]

In the Neurospora circadian system, the White Collar Complex (WCC) formed by WC-1 and WC-2 drives expression of the frequency (frq) gene whose product FRQ feedbacks to inhibit transcriptional activity of WCC. Phosphorylation of WCC has been extensively studied, but the extent and significance of other post-translational modifications (PTM) have been poorly studied. To this end, we used mass-spectrometry to study alkylation sites on WCC, resulting in discovery of nine acetylation sites. Mutagenesis analysis showed most of the acetylation events individually do not play important roles in period determination. Moreover, mutating all the lysines falling in either half of WC-1 or all the lysine residues in WC-2 to arginines did not abolish circadian rhythms. In addition, we also found nine mono-methylation sites on WC-1, but like acetylation, individual ablation of most of the monomethylation events did not result in a significant period change. Taken together, the data here suggest that acetylation or mono-methylation on WCC is not a determinant of the pace of the circadian feedback loop. The finding is consistent with a model in which repression of WCC’s circadian activity is mainly controlled by phosphorylation. Interestingly, light induced expression of some light-responsive genes has been modulated in certain wc-1 acetylation mutants, suggesting that WC-1 acetylation events differentially regulate light responses. [ABSTRACT FROM AUTHOR]

In eukaryotes, repetitive DNA can become silenced de novo, either transcriptionally or post-transcriptionally, by processes independent of strong sequence-specific cues. The mechanistic nature of such processes remains poorly understood. We found that in the fungus Neurospora crassa, de novo initiation of both transcriptional and post-transcriptional silencing was linked to perturbed chromatin, which was produced experimentally by the aberrant activity of transcription factors at the tetO operator array. Transcriptional silencing was mediated by canonical constitutive heterochromatin. On the other hand, post-transcriptional silencing resembled repeat-induced quelling but occurred normally when homologous recombination was inactivated. All silencing of the tetO array was dependent on SAD-6, fungal ortholog of the SWI/SNF chromatin remodeler ATRX (Alpha Thalassemia/Mental Retardation Syndrome X-Linked), which was required to maintain nucleosome occupancy at the perturbed locus. In addition, we found that two other types of sequences (the lacO array and native AT-rich DNA) could also undergo recombination-independent quelling associated with perturbed chromatin. These results suggested a model in which the de novo initiation of transcriptional and post-transcriptional silencing is coupled to the remodeling of perturbed chromatin. [ABSTRACT FROM AUTHOR]

Agarwood is a resinous heartwood of Aquilaria sinensis that is formed in response to mechanical wounding. In the present study pre-treatment of Aquilaria sinensis was carried out, and then the dominant fungi were isolated and purified from the surface and electroshock holes of trees. The isolated Trichoderma sp. and Neurospora sp. were then screened for resistance against benzyl acetone and then inoculated into healthy Aquilaria sinensis trees. After six months, the agarwood was collected for analysis. The chemical composition of incense was analyzed using gas chromatography-mass spectroscopy, and 82 chemical constituents were identified. Agarwood products formed by using Trichoderma sp. and Neurospora sp. consisted of 50.22% and 48.71% ether extracts, respectively, which surpassed the 10% threshold specified by the Chinese Pharmacopoeia. Similarly, relative aromatic contents in the two agarwood products were 30.1% and 32.86%, while proportions of sesquiterpene constituents were 10.21% and 11.19%, respectively. These two agarwood-specific chemical constituents accounted for a large proportion of the total chemical composition, which showed that the generated agarwood was of good quality. The results of the study revealed that both Trichoderma sp. and Neurospora sp. were able to effectively induce agarwood production in Aquilaria sinensis trees in 6 months. This study expands the library of fungi that promote the production of agarwood from Aquilaria sinensis trees. [ABSTRACT FROM AUTHOR]

ABSTRACT Sexual reproduction in fungi allows genetic recombination and increases genetic diversity, allowing adaptation and survival. The velvet complex is a fungal-specific protein assembly that regulates development, pathogenesis, and secondary metabolism in response to environmental cues, such as light. In Neurospora crassa, this complex comprises VE-1, VE-2, and LAE-1. Deletion of ve-1 or ve-2, but not lae-1, leads to increased conidiation (asexual spore formation) and reduced sexual development. Mutants lacking ve-1 and/or ve-2 are female sterile and male fertile, indicating that a VE-1/VE-2 complex regulates the development of female structures. During sexual development, we observed differential regulation of 2,117 genes in dark and 4,364 genes in light between the wild type and the ∆ve-1 strain. The pheromone response and cell wall integrity pathways were downregulated in the ∆ve-1 mutant, especially in light. Additionally, we found reduced levels of both total and phosphorylated MAK-1 and MAK-2 kinases. In vitro experiments demonstrated the binding of VE-1 and VE-2 to the promoters of mak-1 and mak-2, suggesting a direct regulatory role of VE-1/VE-2 in the transcriptional control of MAPK genes to regulate sexual development. Deletion of the photosensor gene white-collar 1 prevented the light-dependent inhibition of sexual development in the ∆ve-1 mutant by increasing transcription of the pheromone response and cell wall integrity pathway genes to the levels in the dark. Our results support the proposal that the regulation of the MAP kinase pathways by the VE-1/VE-2 complex is a key element in transcriptional regulation that occurs during sexual development.IMPORTANCESexual reproduction generates new gene combinations and novel phenotypic traits and facilitates evolution. Induction of sexual development in fungi is often regulated by environmental conditions, such as the presence of light and nutrients. The velvet protein complex coordinates internal cues and environmental signals to regulate development. We have found that VE-1, a component of the velvet complex, regulates transcription during sexual development in the fungus Neurospora crassa. VE-1 regulates the transcription of many genes, including those involved in mitogen-activated protein kinase (MAPK) signaling pathways that are essential in the regulation of sexual development, and regulates the activity of the MAPK pathway. Our findings provide valuable insights into how fungi respond to environmental signals and integrate them into their reproductive processes.

Circadian clocks temporally coordinate daily organismal biology over the 24-h cycle. Their molecular design, preserved between fungi and animals, is based on a core-oscillator composed of a one-step transcriptional-translational-negative-feedback-loop (TTFL). To test whether this evolutionarily conserved TTFL architecture is the only plausible way for achieving a functional circadian clock, we adopted a transcriptional rewiring approach, artificially co-opting regulators of the circadian output pathways into the core-oscillator. Herein we describe one of these semi-synthetic clocks which maintains all basic circadian features but, notably, it also exhibits new attributes such as a "lights-on timer" logic, where clock phase is fixed at the end of the night. Our findings indicate that fundamental circadian properties such as period, phase and temperature compensation are differentially regulated by transcriptional and posttranslational aspects of the clockworks. Synopsis: Eukaryotic circadian oscillators share a one-step transcription-translation negative-feedback loop (TTFL) circuit design, where a main transcriptional complex drives expression of a negative element that can inhibit its own expression. Here, a transcriptional rewiring strategy in the fungus N. crassa reveals that this basic circuit topology can be dramatically changed, while still yielding a functional semi-synthetic clock. Placing the transcription of the negative component frq under the control of the output promoter con-10 alters its regulation, while still yielding rhythmic expression. In the resulting functional semi-synthetic circuit, frq expression is now under the control of circadian clock downstream components. FRQ phosphorylation has a more pronounced effect on circadian period length and temperature compensation than its transcriptional regulation. In the semi-synthetic oscillator, the circadian rhythm phase is now determined by the moment the light is turned on. Co-option of downstream transcription factors into the main transcriptional circuit of the circadian clock in the fungus Neurospora crassa leads to altered clock phase determination. [ABSTRACT FROM AUTHOR]

The application of Neurospora sp., a fungus that commonly thrives on complex agricultural and plant wastes, has proven successful in utilizing citrus peel waste as a source of naringin. A UV-Vis spectrophotometric method proved the biotransformation of naringin, with an absorption maximum (λmax) observed at 310 nm for the biotransformed product, naringenin (NAR). Further verification of the conversion of naringin was provided through thin layer chromatography (TLC). The Neurospora crassa mediated biotransformation of naringin to NAR was utilized for the rapid (within 5 min) synthesis of silver (Ag) and gold (Au) nanoconjugates using sunlight to accelerate the reaction. The synthesized NAR-nano Ag and NAR-nano Au conjugates exhibited monodispersed spherical and spherical as well as polygonal shaped particles, respectively. Both of the nanoconjugates showed average particle sizes of less than 90 nm from TEM analysis. The NAR-Ag and NAR-Au nanoconjugates displayed potential enhancement of the antimicrobial activities, including antibacterial and nematicidal properties over either standalone NAR or Ag or Au NPs. This study reveals the potential of naringinase-producing Neurospora sp. for transforming naringin into NAR. Additionally, the resulting NAR-Ag and NAR-Au nanoconjugates showed promise as sustainable antibiotics and biochemical nematicides. [ABSTRACT FROM AUTHOR]

Circadian clocks drive a large array of physiological and behavioral activities. At the molecular level, circadian clocks are composed of positive and negative elements that form core oscillators generating the basic circadian rhythms. Over the course of the circadian period, circadian negative proteins undergo progressive hyperphosphorylation and eventually degrade, and their stability is finely controlled by complex post-translational pathways, including protein modifications, genetic codon preference, protein–protein interactions, chaperon-dependent conformation maintenance, degradation, etc. The effects of phosphorylation on the stability of circadian clock proteins are crucial for precisely determining protein function and turnover, and it has been proposed that the phosphorylation of core circadian clock proteins is tightly correlated with the circadian period. Nonetheless, recent studies have challenged this view. In this review, we summarize the research progress regarding the function, regulation, and mechanism of protein stability in the circadian clock systems of multiple model organisms, with an emphasis on Neurospora crassa, in which circadian mechanisms have been extensively investigated. Elucidation of the highly complex and dynamic regulation of protein stability in circadian clock networks would greatly benefit the integrated understanding of the function, regulation, and mechanism of protein stability in a wide spectrum of other biological processes. [ABSTRACT FROM AUTHOR]

Meat alternatives are in great demand in current situation to address the increasing human health and animal welfare issues. This study focuses on microbial protein meats, emphasizing the importance of sourcing structurally similar fungal strains and optimizing processing conditions. A new strain, Neurospora intermedia , was identified and evaluated for availability. The impact of pH (3, 5, and 7) and soluble starch (SS) addition (0, 3, and 6%) on the textural and water retention properties of composite mycoprotein gel meat (CMGM) developed by using N. intermedia was investigated. Zeta potential, dynamic rheology, SEM, and FTIR techniques were employed to elucidate the mechanism. CMGM at pH 7 exhibited the sparsest internal structure but demonstrated better elasticity and recovery properties due to strong electrostatic repulsion. Lowering pH enhanced material interaction, improving gel intensity and water-holding capacity after steaming. A 6% SS addition significantly enhanced CMGM's hardness and reduced internal porosity, which further enhanced the water retention, but also reduced fracture elongation. Thus a 3% SS addition was found to be moderate. The mycelium and gel in the system exhibited complementary characteristics. SPI and SS were dispersed under the guidance of the mycelial fiber framework before steaming. The materials were fully crosslinked after steaming and the mycelial fibers were fixed in the gel material, which conferred support to the gel material. As a result, the modulation of excipients can achieve CMGM with different properties—a systematic approach to producing high-quality microbial protein meat. [Display omitted] • Neurospora intermedia ZJU-23 is a suitable strain to prepare meat analogs. • pH and SS addition affected CMGM texture and water-holding properties. • The interactions between mycelium, SS and SPI varied greatly at different pH. • The mycelium and gel in the system exhibited complementary characteristics. [ABSTRACT FROM AUTHOR]

• Neurospora intermedia has the potential to cause an endobronchial mass. • N. intermedia develops orange conidiating colonies composed of septate hyphae. • Coculturing opposite mating types yields ascomata, indicating heterothallism. • N. intermedia cross-reacts with the Aspergillus galactomannan assay in vitro. • N. intermedia is susceptible to amphotericin B and voriconazole. The ascomycete filamentous fungus Neurospora intermedia is commonly used in the food industry and considered nonpathogenic to humans. This study characterizes four N. intermedia isolates recovered from three patients. The first patient had a mediastinal germ cell tumor with multiple metastases. N. intermedia was recovered from his endotracheal aspirate and from the endobronchial mass obtained by bronchoscopic forceps biopsy. Histopathology of the biopsy tissue revealed necrotic tissue mixed with septate fungal hyphae with right-angle branching. An endobronchial mass caused by N. intermedia was thus diagnosed. Another two N. intermedia isolates were recovered from the endotracheal aspirates of two critically ill patients. In vitro, N. intermedia grows rapidly and forms orange, conidiating colonies composed of septate hyphae. Two isolates from the first patient belong to mating type a; the other two isolates belong to mating type A. Coculture of isolates of opposite mating types yielded dark ascomata containing ascospores, supporting that N. intermedia is a heterothallic fungus. N. intermedia isolates cross-reacted with the Aspergillus galactomannan antigen assay and were susceptible to amphotericin B and voriconazole. In conclusion, this report describes the first human infection (endobronchial mass) caused by N. intermedia , highlighting its potential to invade the human respiratory tract. [ABSTRACT FROM AUTHOR]

Sexual reproduction in fungi allows genetic recombination and increases genetic diversity, allowing adaptation and survival. The velvet complex is a fungal-specific protein assembly that regulates development, pathogenesis, and secondary metabolism in response to environmental cues, such as light. In Neurospora crassa , this complex comprises VE-1, VE-2, and LAE-1. Deletion of ve-1 or ve-2 , but not lae-1 , leads to increased conidiation (asexual spore formation) and reduced sexual development. Mutants lacking ve-1 and/or ve-2 are female sterile and male fertile, indicating that a VE-1/VE-2 complex regulates the development of female structures. During sexual development, we observed differential regulation of 2,117 genes in dark and 4,364 genes in light between the wild type and the ∆ ve-1 strain. The pheromone response and cell wall integrity pathways were downregulated in the ∆ ve-1 mutant, especially in light. Additionally, we found reduced levels of both total and phosphorylated MAK-1 and MAK-2 kinases. In vitro experiments demonstrated the binding of VE-1 and VE-2 to the promoters of mak-1 and mak-2 , suggesting a direct regulatory role of VE-1/VE-2 in the transcriptional control of MAPK genes to regulate sexual development. Deletion of the photosensor gene white-collar 1 prevented the light-dependent inhibition of sexual development in the ∆ ve-1 mutant by increasing transcription of the pheromone response and cell wall integrity pathway genes to the levels in the dark. Our results support the proposal that the regulation of the MAP kinase pathways by the VE-1/VE-2 complex is a key element in transcriptional regulation that occurs during sexual development., Importance: Sexual reproduction generates new gene combinations and novel phenotypic traits and facilitates evolution. Induction of sexual development in fungi is often regulated by environmental conditions, such as the presence of light and nutrients. The velvet protein complex coordinates internal cues and environmental signals to regulate development. We have found that VE-1, a component of the velvet complex, regulates transcription during sexual development in the fungus Neurospora crassa . VE-1 regulates the transcription of many genes, including those involved in mitogen-activated protein kinase (MAPK) signaling pathways that are essential in the regulation of sexual development, and regulates the activity of the MAPK pathway. Our findings provide valuable insights into how fungi respond to environmental signals and integrate them into their reproductive processes., Competing Interests: The authors declare no conflict of interest.

Endophytic microbial communities colonize plants growing under various abiotic stress conditions. Candelilla (Euphorbia antisyphilitica Zucc.) is a shrub that develops functionally in arid and semi-arid zones of Mexico; these conditions generate an association between the plant and the microorganisms, contributing to the production of enzymes as a defense mechanism for resistance to abiotic stress. The objective of this research was to isolate and identify endophyte fungi of candelilla and bioprospection of these endophytic fungi for enzyme production using candelilla by-products. Fungi were isolated and identified using ITS1/ITS4 sequencing. Their potency index (PI) was evaluated in producing endoglucanase, xylanase, amylase, and laccase. Fermentation was carried out at 30°C for 8 days at 200 rpm, with measurements every 2 days, using candelilla by-products as substrate. All fungi exhibited higher cellulase, amylase, and laccase activities on the 2nd, 6th, and 8th day of fermentation, respectively, of fermentation. The fungus Aspergillus niger ITD-IN4.1 showed the highest amylase activity (246.84 U/mg), the genus Neurospora showed the highest cellulase activity, reaching up to 13.45 FPU/mg, and the strain Neurospora sp. ITD-IN5.2 showed the highest laccase activity (3.46 U/mg). This work provides the first report on the endophytic diversity of E. antisyphilitica and its potential role in enzyme production., (© 2024 Wiley‐VCH GmbH.)

An investigation on the secondary metabolites from a rice culture broth of the endophytic fungus Neurospora terricola HDF-Br-2 derived from the vulnerable conifer Pseudotsuga gaussenii led to the isolation and characterization of 34 structurally diverse polyketides (1 – 34). Seven of them are previously undescribed, including five unprecedented dihydropyran-containing (terricoxanthones A–E, 1 – 5 , resp.) and one rare tetrahydrofuran-containing (terricoxanthone F, 6) dimeric xanthones. The structures were elucidated by spectroscopic methods and single-crystal X-ray diffraction analyses. Terricoxanthones each were obtained as a racemic mixture. Their plausible biosynthetic relationships were briefly proposed. Compounds 6 , aspergillusone A (8), and alatinone (27) displayed considerable inhibition against Candida albicans with MIC values of 8–16 μg/mL. 4-Hydroxyvertixanthone (12) and 27 exhibited significant inhibitory activities against Staphylococcus aureus , with MIC values of 4–8 μg/mL. Furthermore, compounds 8 and 27 could disrupt biofilm of S. aureus and C. albicans at 128 μg/mL. The findings not only extend the skeletons of xanthone dimers and contribute to the diversity of metabolites of endophytes associated with the endangered Chinese conifer P. gaussenii , but could further reveal the important role of protecting plant species diversity in support of chemical diversity and potential sources of new therapeutics. Five unprecedented dihydropyran-containing and one undescribed tetrahydrofuran-containing dimeric xanthones were isolated and characterized from the endophytic fungus Neurospora terricola HDF-Br-2 associated with the endangered conifer Pseudotsuga gaussenii. The dimers reported herein each were obtained as a racemic mixture. Some isolated xanthones exhibited antimicrobial and antibiofilm effects against Staphylococcus aureus and Candida albicans. [Display omitted] • Six undescribed dimeric xanthones were obtained from an endophytic Neurospora sp. • N. terricola HDF-Br-2 was isolated from the endangered conifer Pseudotsuga gaussenii. • (±)-Terricoxanthones A–E are unprecedented dihydropyran-containing dimeric xanthones. • The xanthone dimers reported herein each were obtained as a racemic mixture. • Some isolated xanthones exhibited antibacterial and antibiofilm effects. [ABSTRACT FROM AUTHOR]

Some cell biological studies of Neurospora crassa have been limited by the rapid rates of hyphal growth and fusion. In this study, we investigated the causative mutation in the standard C24 allele of for (FGSC #9) and assayed the growth and circadian phenotype of the for strain under different nutritional conditions. We show that the for strain can be maintained as metabolically active single cells for 2 days before its growth advances into branched mycelia. This culturing system offers the potential to advance subcellular dynamic research and to facilitate greater understanding of N. crassa in the early developmental stages., Competing Interests: Competing Interests The authors declare no competing interests.